Raman spectroscopy is a well-established method to examine materials by investigating inelastic scattering of probe light. In 1928, C. V. Raman was the first who observed that intense light incident on a sample creates a wavelength shift due to inelastic scattering of light at phonons representing vibrational excitation modes of chemical bonds. The general concept of Raman scattering is explained with reference to the left half of FIG. 1. As indicated therein, an incident photon, also referred to as “pump photon” with a frequency ωpump is incident on a Raman active medium and gets annihilated. The medium which was in a quantum mechanical ground state before gets excited into a vibrational or rotational state, and a scattered photon with a shifted frequency ωemission is generated wherein the difference in frequency ω(or energy ℏω) corresponds to the energy difference in the vibrational/rotational state of the medium. If the frequency of the scattered photon ωemission is smaller than ωpump, it is called a “Stokes photon”, if the frequency is higher, it is called an “anti-Stokes” photon. The frequency or wavelength shifts occurring between the incident and the emitted photons are hence indicative of the vibrational or rotational states of the medium and are consequently highly specific for different media, such as different molecules. In this regard, the Raman spectrum can be regarded as a “fingerprint” of a molecule by which it can be identified.
Raman spectrometers are used as routine analysis tools in many physics and chemistry research facilities. However, due to the small Raman scattering cross-section, pump lasers with Watt level optical output powers are usually required. Only recently, with the introduction of compact and highly efficient high power diode lasers, portable Raman spectrometers for example for drug detection have become available. However, since the Raman signal is inherently weak, for lower sample concentrations the acquisition of a single spectrum usually takes from several seconds up to minutes.
A particularly attractive application of Raman spectroscopy is the so called “Raman microscopy” or “Raman micro-spectroscopy”, as for example described in G. Turrell and J. Corset, eds., Raman microscopy developments and applications (Academic Press, 1996). In these techniques, space resolved Raman spectra are obtained with high resolution, thereby allowing for a very powerful imaging with molecular contrast. Raman microscopy or micro-spectroscopy has been applied for imaging in inorganic and organic samples. Especially for biomedical imaging applications, there is currently a great hope that in the future Raman microscopy may be a potent biomedical imaging modality for in vitro or in vivo microscopy, providing molecular contrast without exogenous contrast agents. However, again the small Raman scattering cross-section makes Raman microscopy prohibitively slow for many applications. In practice, typically imaging protocols are chosen where only a few spectra at certain position on the sample are acquired, rather than a full high resolution en face image. An increase in imaging speed by a factor of 100 to 1000 would be highly desired.
In almost all applications, the weak Raman cross-section is the main problem of Raman spectroscopy and Raman microscopy. Even in cases where a long acquisition time is generally feasible, the small signal levels often make it difficult to identify the Raman signal on the fluorescence background. Various techniques have been proposed to increase the signal level of the detected Raman bands, such as coherent anti-Stokes Raman scattering (CARS), as for example described in A. Zumbusch, et al., “Three-dimensional vibrational imaging by coherent anti-Stokes Raman scattering”, Physical Review Letters 82, 4142-4145 (1999) and C. L. Evans, et al., “Chemical imaging of tissue in vivo with video-rate coherent anti-Stokes Raman scattering microscopy,” Proc Nat Acad Sci 102, 16807-16812 (2005).
A further technique that was proposed for this purpose is the so called surface enhanced Raman scattering (SERS) as for example described in M. G. Albrecht and J. A. Creighton, “ANOMALOUSLY INTENSE RAMAN-SPECTRA OF PYRIDINE AT A SILVER ELECTRODE,” Journal of the American Chemical Society 99, 5215-5217 (1977). In SERS the near-field enhancement effect of the electric field in the proximity of sharp nanostructures on the sub-wavelength scale is used. Plasmonic resonances can further push the field enhancement factor which amplifies both, the electric field of the pump laser and the electric field of the scattered Raman signal. SERS can be cheap and very efficient; however, since it is based on a near-field, in most cases only substances that can be absorbed to a surface can be analyzed. In addition, SERS may provide distorted spectra due to plasmonic resonances of the field enhancing structures.
A further technique to increase the Raman signal intensity is the so called stimulated Raman scattering, which is illustrated on the right hand side of FIG. 1. In stimulated Raman scattering, besides the pump photon with frequency ωpump, a photon with a Stokes frequency ωprobe is incident on the material/probe. The Raman effect leads to an amplification of the probe signal by generating a coherent ray of Stokes photons matching the Raman probe photon. The application of a Raman probe laser greatly enhances the scattering signal but it also introduces a background signal. While stimulated Raman scattering has been known since the 1970s (A. Owyoung, “CW STIMULATED RAMAN-SPECTROSCOPY,” Abstracts of Papers of the American Chemical Society 175, 124-124 (1978)), the lack of low noise, widely and rapidly tunable laser sources in combination with the low signal levels impeded the widespread application.
A system for stimulated Raman spectroscopy, particularly a device for microscopy imaging systems, is described in US 2010/0046039. In this system, a first train of pulses at a first center optical frequency ω1 and a second train of pulses at a second center optical frequency ω2 are provided. The difference between ω1 and ω2 is chosen to be resonant with a specific vibrational frequency of a sample that is to be detected.
A beam property of the second train of pulses, such as its amplitude or its polarization is modulated at a frequency of at least 100 kHz. The first and second trains of pulses are directed toward a common focal volume. Downstream of the sample, the second train of pulses is blocked, and an integrated intensity of substantially all optical frequency components of the first train of pulses transmitted or reflected through the common focal volume is detected. Then, a modulation at the modulation frequency f of the integrated intensity of all optical frequency components of the first train of pulses due to the non-linear interaction of the first train of pulses with the second train of pulses in the common focal volume is provided by means of a lock-in detector, which is indicative of the degree of stimulated Raman emission. The idea of the lock-in detection is to use the modulation frequency to extract the signal from the large background of the signal of the first train of pulses itself. If E0 is the intensity of the signal of the first train of pulses and ΔE is the gain due to stimulated Raman emission, the heterodyning term ΔE·E0 can be extracted from the intensity background E02, because it is modulated at the before mentioned modulation frequency f. By electronically filtering out all contributions of the signal except for the signal at the frequency f using a lock-in detector, it is possible to reject most of the background and to determine the contribution of stimulated emission.
Unfortunately, however, lock-in detectors are rather expensive devices. Accordingly, including the modality of this prior art in a microscope would severely increase its price.
A further major source of costs in ordinary Raman spectroscopy systems is the laser sources, where usually picosecond lasers or femtosecond lasers are employed, which are generally very costly as well.